Field of Invention
The present invention is in the field of medicine and more specifically in the fields of neurosurgery, traumatology and maxillofacial surgery as applied to treatment of peripheral nerve injuries. These injuries are effectively treated with engineered recombinant nucleic acids. One example of such an engineered recombinant nucleic acid is a plasmid that encodes and expresses vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) when contacted with or transformed into a tissue.
Discussion of the Background
About 3-10% of the population sustains peripheral nervous system injuries [1-3]. Peripheral nerve injuries are a common cause of occupational disability and such injuries not only incapacitate numerous workers or working age individuals, but reduce the quality of life. Rehabilitation of a peripheral nerve injury can require a prolonged period of treatment including periods of a year or longer. Photographs of peripheral nervous system injuries and their symptoms are shown by the Figures.
Existing methods for treating peripheral nervous system injuries depend on the extent and nature of the injury in a particular individual including a mechanism of the injury, extent of the peripheral nervous system defect, distance from the location of the injury of the peripheral nerve to the innervated area, and the time elapsed between the injury and surgical intervention.
One type of the reconstructive treatment involves reconnection of the incised nerve ends by means of the end-to-end anastomosis. Peripheral nerve injuries are often accompanied by the formation of prolonged defects, thereby rendering this approach inapplicable. In such cases, autologous nerve grafting is the most appropriate option for repairing prolonged nerve defects. A nerve that is less functionally significant can be used as an autologous graft. Another treatment involves replacement of a peripheral nervous system tissue defect with various structures that create conditions for peripheral nerve regeneration, such as a tubular structure that is designed to replace an extended tissue defect and foster peripheral nerve regeneration. However, despite the advances in reconstructive techniques to restore the peripheral nerve integrity only a partial recovery of the function of an innervated extremity usually occurs even under the most favorable conditions.
These limitations of conventional modes of treating peripheral nervous system injuries necessitate a further search for new methods that enhance results of standard reconstructive treatment, reduce morbidity, disability and generally improve quality of patient's life.
One concept under study involves use of growth factors to induce regeneration of a peripheral nerve. This concept has resulted from the accumulation of knowledge about the roles various growth factors play in the natural process of peripheral nerve health, growth, and regeneration [4].
Vascular endothelial growth factor (VEGF) is one of the well-studied growth factors that affect recovery of peripheral nerves. VEGF is one of the main regulators of angiogenesis and vasculogenesis. It is a disulfide-bound dimeric glycoprotein having an average molecular weight of 34-42 kDa. VEGF-A is a specific mitogen for endothelial cells (ECs) and induces their proliferation, activation, differentiation and formation of EC capillary tubules. These capillary tubules are further remodeled into mature blood vessels. VEGF also induces expression of antiapoptotic proteins and increases survival of ECs. Serious defects and improper development of the cardiovascular system occurs in animals where genes encoding VEGF have been deleted. These defects may be fatal.
A human VEGF is encoded by a gene located on the chromosomal locus 6p21.3. The coding region comprises about 14,000 bps. VEGF has several isoforms including VEGF 121, VEGF 145, VEGF 148, VEGF 165, VEGF 183, VEGF 189, and VEGF 206. These isoforms result from the alternative splicing of VEGF mRNA which consists of 8 exons. Different isoforms of VEGF have biochemical differences in the ability to bind heparin- and heparan-sulphate which permits them to traffic to different extracellular locations. Differences in biochemical properties or extracellular trafficking of human VEGF-A isoforms are attributable to the alternative splicing of exons 6 and 7, because all transcripts of the human VEGF-A gene contain exons 1-5 and 8.
VEGF had long been considered only as an inductor of angiogenesis and as a potential therapeutic agent for treatment of different disorders accompanied by tissue ischemia. However, new data on VEGF's neuroprotective properties for neurons of both the peripheral and central nervous systems have been obtained [5, 6]. VEGF stimulates proliferation of Schwann cells, astrocytes, microglia, and cortical neurons [7-10]. A significant increase of expression of VEGF and Flt-1 (VEGF type II receptor) in the lumbar spine in response to an injury was shown in a rat sciatic nerve crush injury model [11]. The axonal sprouting that manifests as the increased axon number in the conduit per a unit of the cross section area was observed when VEGF was used as a part of the matrigel filling in the conduit [12].
The use of VEGF-loaded poly-lactic acid microspheres in an autologous vein graft in a model of trauma with an extensive defect of fibular and tibial nerves was found to improve the nerve functional index and to increase the number of myelinated fibers in the graft [13].
VEGF has been shown to induce Schwann cell division and migration in a graft towards distal parts that correlates with the increased number of capillaries and myelinated fibers [14].
Introduction of VEGF in combination with a Brain-derived neurotrophic factor (BDNF) into cavernosal bodies in a rat cavernous nerve injury model resulted in the recovery of the lost innervation and erectile function [15].
FGF is another growth factor that induces neurogenesis. FGF induces Schwann cell proliferation and migration in a peripheral nerve injury [16].
In experiments using animal models, it was shown that blocking receptors for FGF, Fgfr1 and Fgfr2, caused neuropathy of non-myelinating sensory fibers and significant impairment of the thermal pain sensitivity [17].
The use of bone marrow-derived stem cells in a peripheral nerve injury model resulted in increased FGF expression that induced migration and proliferation of Schwann cells [18].
In a thoracic spinal cord injury model, the use of FGF in a sciatic nerve graft promoted the improvement of the upper extremity motor function [19].
Therapeutic applications of growth factors, such as VEFG and FGF, were known to have a number of limitations. After the administration into the injury site the growth factors undergo rapid degradation and, therefore, their constant concentration cannot be maintained to achieve the desired therapeutic effect [20].
Gene therapy using vectors that express growth factors like VEGF had previously been performed. There are two main trends in gene therapy: (i) use of viral vectors and (ii) use of non-viral vectors. These different trends generally operate through different mechanisms of gene transfer. The use of viral vectors in the clinical setting, despite their high transfection activity, is limited due to the risk of insertional mutagenesis and potential induction of the inflammatory response and toxicity.
A safer method of gene transfer is based on the use of plasmid DNA. In a model of musculocutaneous nerve repair with end-to-end and end-to-side anastomosis, intraoperative administration of a DNA plasmid comprising a vegf gene into a distal region resulted in the significantly increased number of myelinated fibers per a unit of the cross-section area of the region distal to the anastomosis site that correlated with a significant increase of the VEGF concentration in Schwann cells [21].
A gene-therapeutic construction could be injected paraneurally. In a sciatic nerve injury model, plVEGF was administrated intramuscularly and was combined with a hyaluronic acid film sheath which covered the anastomosis site in order to reduce severity of the scarring. The drug intramuscular injection was accompanied by a significant increase of the muscular response amplitude and the increased number of myelinated fibers distal to the anastomosis site against their use as monotherapy [22].
The study performed by Wang F. et al. demonstrated a plVEGF dose-dependent effect when the gene therapeutic construction was given intraneurally after the end-to-end suturing of sciatic nerve stumps. The use of a higher dosage resulted in the most pronounced increase of neurophysiological parameters and a lesser decrease of the calf muscle weight index [23].
Synergism in action of some factors has been uncovered. For example, combined use of a VEGF gene-coding plasmid and a plasmid encoding the C—CSF gene in a sciatic nerve injury model demonstrated a more pronounced increase in the number of myelinated fibers and capillaries in the region distal to the end-to-end anastomosis, maintenance of more neurons in the spinal ganglia as well as the early recovery of the motor function [24]. However, only a part of the cells is transfected with plasmid DNA when using gene therapeutic agents in vivo. Consequently, the probability that a cell is transfected simultaneously with two different gene therapeutic constructions is reduced. The efficacy of a combination of genetic sequences of two growth factors having a synergistic action in one plasmid has been demonstrated in an animal model of the spinal cord contusion injury.
During this experiment it had been shown that when 40 μg of a VEGF and FGF2 gene containing plasmid were directly injected into the spinal cord, there was a significant increase of the capillary number in the sections made at 1.5 cm from the trauma core. Based on the behavior test data, the recovery of the motor function significantly improved as compared to the control group of animals that were not given the plasmid containing VEGF and FGF2 genes. Based on the results obtained in this experiment, it had been concluded that application of the double cassette plasmid improves spinal cord vascularization and reduces the area of destruction of the spinal gray and white matter [25].
The use of gene therapeutic constructions comprising VEGF and the basic fibroblast growth factor to improve sciatic nerve recovery has been described in [26]. Patent RU 2459630 C1 “Stimulation Technique for Neuroregeneration with Genetic Constructions” describes a method of the post-traumatic regeneration of the rat spinal cord when injecting a double-cassette plasmid pBud-VEGF-FGF2.
Spinal cord and peripheral nerves exhibit significant differences in the regenerative potential. Consequently, the results described above have given no indication as to whether the treatment of a peripheral nerve injury with a plasmid expressing both VEGF and FGF2 could be effective in repairing peripheral nerve injuries. This is due to the fact that the mechanism of a contusion injury significantly differs by pathogenesis and a degree of severity from a trauma accompanied with neurotmesis which is more specific for peripheral nerves and prevails in the total structure of their injuries. Moreover, as mentioned above, the type or extent of regenerative or recuperative effects of applying particular growth factors, or a combination of growth factors, to damaged peripheral nervous system tissue are substantially unexplored.
Keeping in mind these problems with protein-based therapies and uncertainties regarding responsiveness of peripheral nerve injuries to a nucleic-acid based therapy, the inventors have developed nucleic-acid based vectors that express growth factors such as VEGF and FGF2 and initiated studies to determine whether incorporation of these growth factors into a complex therapy of a peripheral nerve repair could be effective. As shown herein, a better, more reliable, and more effective treatment of peripheral nerve injuries is possible using a nucleic acid-based therapy.